Electrophoresis is a well-known technique for the separation of charged species by utilizing their differences in rate of migration under the influence of an electrical field. The prototype of all modern electrophoretic methods is free, or moving-boundary, electrophoresis. The mobility .mu. in square centimeters per volt-second of a molecule in an electric field is given by the ratio of the velocity of migration v, in centimeters per second, to electric field strength E, in volts per centimeter: .mu.=v/E. For small ions, such as chloride, .mu. is between 4 and 9.times.10.sup.-4 cm.sup.2 v.sup.-1 s.sup.-1 (25.degree. C.); for proteins, it is about 0.1 to 1.0.times.10.sup.-4 cm.sup.2 v.sup.-1 s.sup.-1. Protein thus migrates much more slowly in an electrical field than small ions simply because they have a much smaller ratio of charge to mass.
Free electrophoresis has been largely supplanted by various forms of zone electrophoresis in which the aqueous protein solution is immobilized in a solid matrix that provides mechanical rigidity and reduces convection and vibration disturbances. Matrix material that is porous also allows for sieving. This form of zone electrophoresis can separate a protein mixture on the basis of both electric charge and molecular size, thereby providing high resolution.
Capillary zone electrophoresis ("CZE") in small bore capillaries was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of certain small solutes J. Chromatog., 218 (1981), page 209; Anal. Chem., 53 (1981) page 1298. Attractive factors for CZE include the small sample sizes, little or no sample pretreatment, high resolution, automation, and the potential for quantification and recovery of biologically active samples. For example, U.S. Pat. No. 4,675,300, inventors Zare et al., issued Jun. 23, 1987 describes theories and equipment for electrokinetic separation processes employing a laser-excited fluorescence detector. The system described by Zare et al. includes a fused silica capillary with a 75.mu. inner diameter.
Unfortunately, one of the single greatest disadvantages of capillary zone electrophoresis lies when attempts are made to separate macromolecules such as proteins. Separations of macromolecules by CZE leads to untoward interactions of the biopolymers with the silica capillary wall.
Jorgenson and Lukacs had noted that separation of model proteins, such as cytochrome, lysozyme and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was accompanied by strong tailing, and suggested this might be caused by Coulombic interactions of the positively charged proteins and the negatively charged capillary wall. Jorgensen et al., Science, 222 (1983) pages 266-272. The authors reported investigating Teflon capillaries but found these to also exhibit significant adsorptivity toward proteins. They attempted to deactivate the surface of fused silica with groups such as trimethyl silane, octadecylsilane, aminopropylsilane, and cross-linked methyl cellulose, which apparently did not succeed. They then turned to bonding glycol-containing groups to the surface.
Lauer and McManigill, Analytical Chemistry, 58 (1986), page 166, have reported that the Coulombic repulsion between proteins and the capillary wall of silica capillaries can overcome adsorption tendencies of the proteins with the capillary wall. They demonstrated separations of model proteins (ranging in molecular weight from 13,000 to 77,000) by varying the solution pH relative to the isoelectric point (pI) of the proteins to change their net charge. However, disadvantages of this approach are that silica begins to dissolve above pH 7, which shortens column life and degrades performance, only proteins with pI's less than the buffer pH can be analyzed, which drastically reduces the range of useful analysis, and interactions which are not Coulombic may still occur even with proteins bearing a net negative charge due to the complexity of protein composition and structure.
Another approach to the problem of biopolymer, or protein, interactions has been to increase ionic strength. It has been demonstrated that this concept works in principle, but heating is also increased as ionic strength is increased. This heating tends to degrade the efficiency of separation.
Yet another approach to the problem of undesirable protein interactions with the capillary wall has been to coat the electrophoresis tube with a monomolecular layer of non-crosslinked polymer. Thus, U.S. Pat. No. 4,680,201, inventor Hjerten, issued Jul. 14, 1987 describes a method for preparing a thin-wall, narrow-bore capillary tube for electrophoretic separations by use of a bifunctional compound in which one group reacts specifically with the glass wall and the other with a monomer taking part in a polymerization process. This free-radical procedure results in a polymer coating, such as polyacrylamide coating, and is suggested for use in coating other polymers, such as poly(vinyl alcohol) and poly(vinylpyrrolidone). However, this method and capillary tube treatment tends to destroy the electroosmotic flow, and efficiencies are still rather low. These rather low efficiencies suggest that undesirable protein-wall interactions are still occurring.
U.S. Pat. No. 4,690,749, issued Sep. 1, 1987, Van Alstine et al., discloses activating a glass surface with aminosilanes to alter reactive surface groups from silanols to amines. Van Alstine et al. teach use of neutral polymers, such as polyethylene glycol or dextran polymer, to be covalently bound to the electrophoretic surface. The data presented by Van Alstine et al. show their treatment reduces electroosmotic flow, which is consistent with their view that electroosmotic flow is not desirable.